Composite

Part:BBa_M36983:Design

Designed by: Nathan Barnett, Meghan Bowler, Kian Torabian   Group: Stanford BIOE44 - S11   (2011-05-17)

Type II Secreted Antifreeze Actuator in E. coli (5-Repeat) Correct


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 107
    Illegal PstI site found at 154
    Illegal PstI site found at 181
    Illegal PstI site found at 199
    Illegal PstI site found at 211
    Illegal PstI site found at 247
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 107
    Illegal PstI site found at 154
    Illegal PstI site found at 181
    Illegal PstI site found at 199
    Illegal PstI site found at 211
    Illegal PstI site found at 247
    Illegal NotI site found at 282
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 311
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 107
    Illegal PstI site found at 154
    Illegal PstI site found at 181
    Illegal PstI site found at 199
    Illegal PstI site found at 211
    Illegal PstI site found at 247
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 107
    Illegal PstI site found at 154
    Illegal PstI site found at 181
    Illegal PstI site found at 199
    Illegal PstI site found at 211
    Illegal PstI site found at 247
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This composite part consists of a strong 5' UTR Sequence, followed by a signal peptide for secretion from E.coli, the non-glycolated AFP 5-mer, and finally a strong termination sequence. The 5' UTR and termination sequences are very strong because we want high transcription and only transcription of the region we coded before the termination sequence. The signal peptide is N-terminal and is native to E.coli.


Source

Pseudopleuronecta americanus (White Flounder) and E. Coli

References

1. Antifreeze Protien Sequence Information: Solomon, R. G., and Appels, R. (1999) "Stable, High-Level Expression of a Type I Antifreeze Protein in Escherichia Coli." Protein Expression and Purification 16: 53-62.

2. Secondary Structure Prediction Tool: Jones, David T., Daniel Buchan, Sean Ward, Anna Lobley, Tim Nugent, and Kevin Bryson.PSIPRED: Protein Structure Prediction Server. PSIPRED. BioInformatics Group - University College London, Oct. 2010. Web. Apr. 2011. <http://bioinf.cs.ucl.ac.uk/psipred/>.

3. Codon Optimization Tool: Richardson, S. M., S. J. Wheelan, and J. D. Boeke. "GeneDesign: Codon Juggling." The Bader Lab. Dec. 2009. Web. Apr. 2011. <http://genedesign.thruhere.net/cgi-bin/gd/gdCodJug.cgi>.

4. Protein Secretion: (SEC pathway): Oudega, B. (2003). Protein secretion pathways in bacteria. Dordrecht: Kluwer Academic Publishers.

5. Signal Peptide Sequence: Gennity, J., Goldstein, J., and Inouye, M. (1989) “Signal Peptide Mutants of Escherichia coli.” Journal of Bioenergetics and Biomembranes 22(3):233-269

6. Used to see if modified signal peptide could be predicted after codon optimization: Hiller K, Grote A, Scheer M, Munch R, Jahn D. (2004) “PrediSi: prediction of signal peptides and their cleavage positions.” Nucleic Acids Res 32:W375–379.